The goal of this project is to understand in mechanistic terms how proteins are transported across membranes. Previous work has demonstrated that secretory and membrane proteins are translocated from the cytosol across the membrane through a channel formed from a membrane protein complex, called the Sec61 p complex in eukaryotes and the SecY complex in bacteria and archae. The recently determined the Xray structure of an archaeal SecY complex provides the basis for the present proposal. In eukaryotes, there is a translocation pathway in the reverse direction, called ERAD (for ER associated degradation), by which misfolded ER proteins are degraded by the proteasome. Our recent experiments have led to the identification of most, if not all, ERAD components, which pave the way for mechanistic studies. Here, we will address key aspects of translocation with specific emphasis on the following questions: 1. How does the protein-conducting channel bind to the ribosome and how is it gated? We will use electron cryo-microscopy to elucidate how the channel binds the ribosome during co- translational translocation. We will investigate how the channel is opened and how the membrane barrier for small molecules is maintained during protein translocation. 2. What is the role of SecY complex oligomers and how does SecA move polypeptides through the channel? We will clarify the role of oligomers of SecY complexes in translocation, investigate how SecA binds to the SecY channel, and determine how SecA moves polypeptide chains through the channel. 3. Are there different ERAD pathways? We will test the existence of different ERAD pathways (called ERAD-L, -M, and -C), depending on whether the misfolded domain of a protein is located in the lumen, membrane, or cytosol, by screening for substrates in S. cerevisiae. We will test the temporal order of translocation and poly-ubiquitination, and determine whether one pathway is dominant over another. 4. What is the molecular mechanism of ERAD? We will establish assays that recapitulate sub-reactions of ERAD with purified yeast components with the goal to develop a system that mimics retro-translocation in detergent. Mutants in ERAD components will be employed to elucidate the mechanism of ERAD-L, and crosslinking methods will be used to identify candidate proteins that may form the retro-translocation channel. The mechanism of protein translocation is of great medical significance. A large number of diseases are known in which mutations cause the misfolding of proteins in the ER and their subsequent degradation in the cytosol. Examples include cystic fibrosis and a1-antitrypsin deficiency. The pathway is also hijacked by certain viruses and toxins, and a better understanding may lead to new drugs allowing interference.